Derstanding and predicting the evolvability of drug resistance, e.g., the acquisition of antibiotic resistance in a step-wise manner.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptRESULTSHeterogeneous responses to antibiotics Antibiotic susceptibility is typically assayed by counting the colonies formed following bacteria are spread onto agar plates containing several concentrations of antibiotics (21). If these cells exhibit growth bistability, then only the increasing fraction from the inoculant cells will kind colonies. To test for this heterogeneous response, we characterized the fraction of colonies formed by many strains of E. coli expanding on agar within the presence of chloramphenicol (Cm), among the oldest and most-studied translation-inhibiting antibiotics (22). We studied strains that express the Cm-resistance enzyme chloramphenicol acetyltransferase (CAT), which modifies and deactivates Cm as outlined by wellcharacterized biochemistry (23). CAT enzymes are expressed constitutively in our strains, just as they (and quite a few other drug-resistance enzymes and pumps) are generally identified inside the wild (247). Overnight incubation of CAT-expressing strains on Cm-agar plates revealed indicators of population-level heterogeneity. For 1 such strain, Cat1 (table S1), the amount of colonyforming units (CFU) decreased gradually on plates with escalating Cm concentrations (Fig. 1A, best; fig. S2B). Hence, only a fraction with the plated cells formed visible colonies (Fig. 1B, circles), even at concentrations properly beneath the empirical minimal inhibitory concentration at which colony formation is completely inhibited (MICplate, fig. S2A). It can be unlikely that heterogeneity arose from spontaneous mutation, as repeating the experiment utilizing a single colony isolated at 90 MICplate developed qualitatively related final results (with CFU decreasing at intermediate drug levels, fig. S2C ). In contrast, CFU count of CAT-less wild type cells (strain EQ4) remained higher till comprehensive inhibition at MICplate (Fig. 1A bottom; fig. S3), indicating that the vast majority of plated cells grew up to the MIC (Fig. 1B, triangles). Direct observation of Reverse Transcriptase Inhibitor Purity & Documentation development bistability by microscopy To confirm the SHP2 drug coexistence of developing and non-growing cells directly, we employed a microfluidic device in which the development of individual (immotile) cells could possibly be tracked with time-lapse microscopy for extended periods (28) as they grew inside the presence of Cm. The device gives a steady provide of fresh media to several development chambers, whose heights are adjusted to be slightly bigger than the width of a single bacterium ( 1 m), enabling cells to develop for up to 9 generations into monolayer colonies in every single chamber (fig. S4). Immotile CAT-expressing cells (Cat1m) expanding exponentially in Cm-free batch culture have been transferred to the microfluidic device, and had been allowed to continue developing exponentially for many generations ahead of switching to growth medium with Cm (see Strategies). With 0.9 mM Cm (90 of MICplate) within the medium, 70 with the cells stopped developing; nongrowing and growing cells had been normally observed side by side in the very same chamber (Fig. 2A, Movie S1). Ultimately, it became not possible to track these non-growing cells that were adjacent to growing populations due to overcrowding. By tracking some non-growing cellsScience. Author manuscript; available in PMC 2014 June 16.Deris et al.Pagethat have been far away from developing populations, we observed that this development bimodality persisted.
